Summary At this time, there is no cure for metastatic breast cancer. The prognosis for these patients is poor. With current standard of care treatments, the median survival time is three years. We propose to develop a novel bacterial drug delivery therapy into an effective and low toxicity therapeutic for breast cancer metastases, that targets protein phosphatase 1 (PP1). Aberrant phosphorylation in cell signaling is a hallmark of cancer. PP1 is one of the major cellular phosphatases and gains its specificity via its interaction with over 200 binding partners. Targeting phosphatases is difficult, due to the difficulty of interrupting protein-protein interactions and the lack of a binding pocket for small molecules. NIPP1 is a potent and specific nuclear inhibitor of PP1. A two-fold increase of NIPP1 blocks PP1 from forming other holo-enzymes and inhibits tumor proliferation. Systemic delivery of this protein drug would induce severe side effects, due to the essential role of PP1. The specific tropism of Salmonella to breast cancer metastases, in ratios of 100,000 to 1 compared to healthy tissue can produce and deliver protein drugs with high specificity to cancer cells and with minimal side effects. The proof of concept for this study has been established by demonstrating that 1) a bacterial lysis system can deliver proteins specifically into the cytoplasm of cancer cells and 2) bacterial delivery of NIPP1 induces cell death in breast cancer cells. In this proposal, we want to determine the safety and efficacy of bacterially delivered NIPP1 (EBT-001) in mice and develop a bacterial delivery strain that is genetically stable and resistant to mutational loss-of-function. In Aim 1, we will test the efficacy of EBT-001 in liver, lung and bone metastases in CD34+ humanized mice. In Aim 2, we will determine the maximum tolerated dose (MTD) of EBT-001 and study acute toxicity in a dose-escalation study. We will measure the biodistribution of this therapy in mice with and without tumors to determine off-target bacterial accumulation and protein delivery. In Aim 3, we will develop a bacterial delivery strain with a constant lysis rate. The leakage of the lysin E gene, when unexpressed, results in bacterial stress and acquired resistance to lysis. A directional promoter switch will be integrated that eliminates leakage in the OFF state and fully activates the lysin E gene in the ON state. This genetic circuit prevents sublethal levels of lysine E in bacteria. The robustness of this system will be tested in culture and in subcutaneous tumors in mice. The successful outcome of this proposal is the first step in the development of EBT-001 as a new therapy for metastatic breast cancer. Further development of this therapy will be pursued in a Phase II SBIR to obtain the necessary data for an IND application. This research will establish a platform technology to directly deliver polypeptides into cells at critical points in signaling pathways and will be effective in a wide array of cancers.